Linescan microscopy data to extract diffusion coefficient of a fluorescent species using a commercial confocal microscope

We report here on the measurement of the diffusion coefficient of fluorescent species using a commercial microscope possessing a resonant scanner. Sequential linescans with a rate of up to 12 kHz yield a temporal resolution of 83 μs, making the setup amenable to measure diffusion rates over a range covering at least three orders of magnitude, from 100 μm(2)/s down to 0.1 μm(2)/s. We share representative data sets covering (i) the diffusion of a dye molecule, observed in media of different viscosities and (ii) the diffusion of a prototypical membrane receptor. The data can be valuable for researchers interested in the rapid diffusion properties of nuclear, cytosolic or membrane bound proteins fused to fluorescent tags

Visualization of class A GPCR oligomerization by image-based fluorescence fluctuation spectroscopy

G protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors conveying extracellular information into intracellular signals. Many GPCRs have been shown to be able to oligomerize and it is firmly established that Class C GPCRs (e.g. metabotropic glutamate receptors) function as obligate dimers. However, the oligomerization capability of the larger Class A GPCRs (e.g. comprising the β-adrenergic receptors (β-ARs)) is still, despite decades of research, highly debated. Here we assess the oligomerization behavior of three prototypical Class A GPCRs, the β1-ARs, β2-ARs, and muscarinic M2Rs in single, intact cells. We combine two image correlation spectroscopy methods based on molecular brightness, i.e. the analysis of fluorescence fluctuations over space and over time, and thereby provide an assay able to robustly and precisely quantify the degree of oligomerization of GPCRs. In addition, we provide a comparison between two labelling strategies, namely C-terminally-attached fluorescent proteins and N-terminally-attached SNAP-tags, in order to rule out effects arising from potential fluorescent protein-driven oligomerization. The degree of GPCR oligomerization is expressed with respect to a set of previously reported as well as newly established monomeric or dimeric control constructs. Our data reveal that all three prototypical GPRCs studied display, under unstimulated conditions, a prevalently monomeric fingerprint. Only the β2-AR shows a slight degree of oligomerization. From a methodological point of view, our study suggests three key aspects. First, the combination of two image correlation spectroscopy methods allows addressing cells transiently expressing high concentrations of membrane receptors, far from the single molecule regime, at a density where the kinetic equilibrium should favor dimers and higher-order oligomers. Second, our methodological approach, allows to selectively target cell membrane regions devoid of artificial oligomerization hot-spots (such as vesicles). Third, our data suggest that the β1-AR appears to be a superior monomeric control than the widely used membrane protein CD86. Taken together, we suggest that our combined image correlation spectroscopy method is a powerful approach to assess the oligomerization behavior of GPCRs in intact cells at high expression levels

A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics

G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the α-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning β-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands

Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone receptor 1

Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to G(s) and increased activation of G(q) compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling

Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone 1 receptor

Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to G(s) and increased activation of G(q) compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling

Molecular details of dimerization kinetics reveal negligible populations of transient µ-opioid receptor homodimers at physiological concentrations.

Various experimental and computational techniques have been employed over the past decade to provide structural and thermodynamic insights into G Protein-Coupled Receptor (GPCR) dimerization. Here, we use multiple microsecond-long, coarse-grained, biased and unbiased molecular dynamics simulations (a total of ~4 milliseconds) combined with multi-ensemble Markov state models to elucidate the kinetics of homodimerization of a prototypic GPCR, the µ-opioid receptor (MOR), embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol lipid bilayer. Analysis of these computations identifies kinetically distinct macrostates comprising several different short-lived dimeric configurations of either inactive or activated MOR. Calculated kinetic rates and fractions of dimers at different MOR concentrations suggest a negligible population of MOR homodimers at physiological concentrations, which is supported by acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments. This study provides a rigorous, quantitative explanation for some conflicting experimental data on GPCR oligomerization

Chiral nonperturvative approach to the isoscalar s-wave pion-pion interaction in a nuclear medium

The s-wave isoscalar amplitude for pion-pion scattering in a nuclear medium is evaluated using a nonperturbative unitary coupled channels method and the standard chiral Lagrangians. The method has proved successful to describe the pion-pion properties in the scalar isoscalar channel up to 1.2 GeV giving rise to poles in the t matrix for the f0(980) and the sigma. The extension of the method to the nuclear medium implies not only the renormalization of the pions in the medium, but also the introduction of interaction terms related to contact terms in the pion-nucleon to pion-pion-nucleon interaction. Off shell effects are also shown to be important leading to cancellations which reduce the coupled channel integral equations to a set of algebraic equations. As the density increases we find a reduction of strength below the $\sigma$ region and a certain accumulation of strength at energies around pion threshold. Our results, based on chiral Lagrangians, provide similar results to those obtained with phenomenological models which impose minimal chiral constraints.Comment: 18 pages, LaTeX, 8 PostScript figures.(Revised version Aug-98, with corrected results and new references

Development of a conformational histamine H(3) receptor biosensor for the synchronous screening of agonists and inverse agonists

The histamine H(3) receptor (H(3)R) represents a highly attractive drug target for the treatment of various central nervous system disorders, but the discovery of novel H(3)R targeting compounds relies on the assessment of highly amplified intracellular signaling events that do not only reflect H(3)R modulation and carry the risk of high false-positive and -negative screening rates. To address these limitations, we designed an intramolecular H(3)R biosensor based on the principle of bioluminescence resonance energy transfer (BRET) that reports the receptor's real-time conformational dynamics and provides an advanced tool to screen for both H(3)R agonists and inverse agonists in a live cell screening-compatible assay format. This conformational G-protein-coupled receptor (GPCR) sensor allowed us to characterize the pharmacological properties of known and new H(3) receptor ligands with unprecedented accuracy. Interestingly, we found that one newly developed H(3) receptor ligand possesses even stronger inverse agonistic activity than reference H(3)R inverse agonists including the current gold standard pitolisant. Taken together, we describe here the design and validation of the first screening-compatible H(3)R conformational biosensor that will aid in the discovery of novel H(3)R ligands and can be employed to gain deeper insights into the (in-)activation mechanism of this highly attractive drug target

Stepwise activation of a class C GPCR begins with millisecond dimer rearrangement

G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the "light receptor" rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within ~1 ms, while all other receptors are thought to become activated within ~50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of τ(1) ~ 1-2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (τ(2) ~ 20 ms). Receptor deactivation occurred with time constants of ~40 and ~900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including-in addition to the inactive and active states-at least two metastable intermediate states

Unmasking features of the auto-epitope essential for β(1)-adrenoceptor activation by autoantibodies in chronic heart failure

AIMS: Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac β(1)-adrenoceptors. Allosteric receptor activation depends on conformational features of the autoantibody binding site. Elucidating these features will pave the way for the development of specific diagnostics and therapeutics. Our aim was (i) to fine-map the conformational epitope within the second extracellular loop of the human β(1)-adrenoceptor (β(1) EC(II)) that is targeted by stimulating β(1)-receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope. METHODS AND RESULTS: Non-conserved amino acids within the β(1) ECII loop (compared with the amino acids constituting the ECII loop of the β(2)-adrenoceptor) were one by one replaced with alanine; potential intra-loop disulfide bridges were probed by cysteine-serine exchanges. Effects on antibody binding and allosteric receptor activation were assessed (i) by (auto)antibody neutralization using cyclopeptides mimicking β(1) ECII ± the above replacements, and (ii) by (auto)antibody stimulation of human β(1)-adrenoceptors bearing corresponding point mutations. With the use of stimulating β(1)-receptor (auto)antibodies raised in mice, rats, or rabbits and isolated from exemplary dilated cardiomyopathy patients, our series of experiments unmasked two features of the β(1) ECII loop essential for (auto)antibody binding and allosteric receptor activation: (i) the NDPK(211-214) motif and (ii) the intra-loop disulfide bond C(209)↔C(215). Of note, aberrant intra-loop disulfide bond C(209)↔C(216) almost fully disrupted the functional auto-epitope in cyclopeptides. CONCLUSIONS: The conformational auto-epitope targeted by cardio-pathogenic β(1)-receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the β(1) EC(II) loop bearing the NDPK(211-214) motif and the C(209)↔C(215) bridge while lacking cysteine C(216). Such molecules provide promising tools for novel diagnostic and therapeutic approaches in β(1)-autoantibody-positive CHF